1. Field of the Invention
This invention relates to a fermentation method to produce succinic acid, and more particularly this invention relates to a method for creating a bacterial strain capable of utilizing a myriad of sugars to produce succinic acid as a major fermentation product.
2. Background of the Invention
Carboxylic acids hold promise as potential precursors for numerous chemicals. For example, succinic acid can serve as a feedstock for such plastic precursors as 1,4 butanediol (BDO) tetrahydrofuran, and gamma-butyroactone. New products derived from succinic acid are under development, with the most notable of these being polyester which is made by linking succinic acid and BDO. Generally, esters of succinic acid have the potential of being new, xe2x80x9cgreenxe2x80x9d solvents that can supplant more harmful solvents. In total, succinic acid could serve as a precursor for millions of pounds of chemicals annually at a total market value of over $1 billion. Along with succinic acid, other 4-carbon dicarboxylic acids, such as malic acid, and fumaric acid also have feedstock potential.
The production of these carboxylic acids from renewable feedstocks (in this case through fermentation processes) is an avenue to supplant the more energy intensive methods of deriving such acids from nonrenewable sources. Succinate is an intermediate for anaerobic fermentations by propionate-producing bacteria but those processes result in low yields and concentrations.
Anaerobic rumen bacteria, such as Bacteroides ruminicola and Bacteroides amylophilus also produce succinate. However, rumen organisms are characteristically unstable in fermentation processes.
It has been long been known that a mixture of acids are produced from E. coli fermentation, as elaborated in Stokes, J. L. 1949 xe2x80x9cFermentation of glucose by suspensions of Escherichia colixe2x80x9d J. Bacteriol. 57:147-158. However, for each mole of glucose fermented, only 1.2 moles of formic acid, 0.1-0.2 moles of lactic acid, and 0.3-0.4 moles of succinic acid are produced. As such, efforts to produce carboxylic acids fermentatively have resulted in relatively large amounts of growth substrates, such as glucose, not being converted to the desired product.
Some bacteria, such as A. succiniciproducens, utilized in fermentation processes as outlined in U.S. Pat. No. 5,143,834 to Glassner et al., naturally produce succinic acid in moderate liters up to only about 35-40 grams per liter (g/L). The A. succiniciproducens host strain has been shown to be not highly osmotolerant in that it does not tolerate high concentrations of salts and is further inhibited by moderate concentrations of product. Lastly, A. succiniciproducens presents handling in that as an obligate anaerobe, procedures using the organism must be done in the absence of oxygen. Also, medium preparation for the inoculum requires the addition of tryptophan.
Previous efforts by the inventors to produce succinic acid has resulted in the isolation and utilization of a mutant bacterium. The mutant, available as ATCC accession number 202021, is the subject of U.S. Pat. Reissue application Ser. No. 09/429,693. Reissue application Ser. No. 09/429,693, incorporated herein by reference, teaches a succinic acid-producing bacterial stain (AFP 111) which spontaneously mutates from its precursor. The mutant is able to grow fermentatively on glucose to produce succinic acid in high yields, while its precursors are unable to do so. However, an obvious drawback to utilizing this method of succinic acid production is its limitation to a single mutant.
Other efforts (U.S. Pat. No. 6,159,738) by the inventors have resulted in a method for constructing bacterial strains having increased succinic acid production. The method teaches that alteration of the phosphotransferase gene of E. coli causes the bacteria to produce more succinic acid. A drawback to this method is its limitation to a single alteration.
A need exists in the art for a method for producing succinic acid fermentatively, whereby the method is not relegated to a single mutant or gene. The method should be enabled by any organism having a particular, and easily determined, genotype. The method should be able to be performed in relatively inert conditions using robust organisms (i.e., those having high feed back inhibition thresholds), and also so as to obviate the need for sophisticated environmental control measures. The method should produce superior results utilizing mixtures of sugars derived from hydrolysis of lignocellulosic materials, inasmuch as these substrates offer a cheaper source of sugars, and as such, their use could reduce production costs for succinic acid.
It is an object of the present invention to provide a method of producing succinic acid that overcomes many of the disadvantages of the prior art.
It is another object of the present invention to provide a fermentation process that produces high yields of succinic acid. A feature of the invention is the utilization of bacterial genomes containing a plurality of mutant genes to enable the method. An advantage of the invention is that bacteria can be readily manipulated to produce the plurality of mutants. Alternatively, bacteria already containing the plurality of mutations can be utilized without further manipulation.
Still another object of the present invention is to provide a process for manipulating bacteria to produce large amounts of succinic acid. A feature of the invention is the disruption of the normal regulation of sugar metabolism in the bacteria. An advantage of the invention is the ability to manipulate a variety of bacteria to facilitate relatively high product-to-growth substrate ratios (i.e., at or above 1:1) in fermentation processes for producing succinic acid. Another advantage of the invention is the ability to utilize bacteria which become glucose metabolisers and non-glucose metabolisers.
Yet another object of the present invention is to produce succinic acid fermentatively. A feature of the invention is the utilization of bacteria containing altered phosphotransferase (pts) systems, pyruvate formate lyase (pfl) systems, and lactate dehydrogenase (ldh) systems. An advantage of the invention is that the bacteria can be derived from many genera which use these enzyme systems for sugar fermentation.
Briefly a method of producing succinic acid from industrial-grade hydrolysates is provided, comprising: supplying an organism that contains mutations for the genes ptsG, pflB, and ldhA; allowing said organism to accumulate biomass; and allowing said organism to metabolize the hydrolysate.
Also provided is a bacteria mutant characterized in that it produces succinic acid from substrate contained in industrial-grade hydrolysate in a ratio of between 0.6:1 and 1.3:1 succinic acid to substrate (e.g., between 0.6 and 1.3 grams succinic acid per gram of total sugar consumed).